The discrimination of particles is useful in numerous clinical assays including ascertaining the types and numerical quantity of cells in blood, ascertaining invasive particles in a fluid sample, such as bacteria and virus, and quantifying the density and volume of cells in a fluid sample.
One method of the above is disclosed in U.S. Pat. No. 5,017,497 issued to de Grooth et al. FIG. 1 of the present application illustrates the apparatus disclosed '497 Patent. Referring to FIG. 1, the '497 Patent discloses a flow cell 2 through which cells from, for example, blood or the like, flow substantially one by one therethrough. A laser input 4 emits a polarized beam of laser light that is oriented substantially orthogonally to the flow of blood cell through flow cell 2 such that the polarized laser light impinges upon the blood cells as they pass through flow cell 2. By "polarized" it is meant that the plane of the electric field oscillation of the laser light is uniform. An optical lens 6 has an aperture which limits the cone of scattered light from the blood cells that can be collected to 72.degree. or less. The central axis of the cone of lens 6 is 90.degree. to both the path of the polarized laser light and the flow of blood cells through flow cell 2. The scattered light emanating from lens 6 is collimated in a matter known in the art. The scattered light now has a mixed polarization that is characteristic of the cell type. The light next passes through a beam splitter 8 that divides the light into two separate beams. A first light beam, substantially concentric with the light beam that originally emanated from lens 6, passes through first polarization analyzer 10. Polarization analyzer 10 is configured to pass therethrough only polarized light having a vector the same as the original laser light. The second beam emanating from beam splitter 8 is oriented substantially perpendicular to the orientation of the first beam emanating from beam splitter 8. This second beam enters second polarization analyzer 12. Second polarization analyzer 12 is configured to pass therethrough only light having a polarization vector substantially orthogonal to the polarization vector of the other beam from beam splitter 8 that passed through first polarization analyzer 10. The beams that pass through first polarization analyzer 10 and second polarization 12 enter polarized detector 14 and depolarized light detector 16, respectively. The ratio of the outputs of polarized light detector 14 and depolarized light detector 16, based on intensity, provide the depolarization ratio.
As shown in FIG. 4 eosinophils, a subset of leukocytes (white blood cells), depolarize the right angle of scattered light quantified by the above configuration to a greater degree than other leukocytes. FIG. 4 is a graphical representation having the output of polarized light detector 14 as one axis and the output of depolarized light detector 16 as the axis . While the above invention does provide some useful data regarding leukocytes, and more specifically eosinophils, as shown in FIGS. 6B, 7B, 7B and 9B, the cluster points within the eosinophil cluster (the cluster points above the angled threshold line on the graphical representation having "DEPOL" as one axis and "ORTHAGONAL" as the other axis) are quite condensed. The dense nature of the points within the eosinophil cluster results in difficulty for the computer software programs that ascertain and identify clusters to accurately identify eosinophil clusters. Additionally, this prior art configuration requires expensive optical devices such as photo multiplier tubes, and lens 6, first polarization amplifier 10 and second polarization amplifier 12.
A need thus exists for a flow cytometer apparatus and related method in which the cell cluster points are less dense for ease of characterization of the different cell clusters. A need also exists for the above apparatus and method which has fewer and less expensive components.